Expression Analysis Of Tea Plant MiR164a And Its Target Gene CsNAC1 And Study Of MiR164a Function

Plant miR164 is involved in complex physiological processes such as plant growth regulation and stress by negatively regulating the target gene NAC transcription factors(NAC transcription factors).In this paper,tea plant miR164 a and its target gene CsNAC1 were isolated.On the basis of analyzing the characteristics of the genetic structure,the expression patterns of the two genes were analyzed,and their function was verified by transgenic technology,providing reference for future resistance molecular breeding of tea plants.The main results are as follows:(1)Obtaining gene sequences and bioinformatics analysis6 members of the miR164 family were obtained from low-temperature miRNA high-throughput sequencing data of tea plant.Designing specific primers for amplification based on the precursor sequence of Csn-miR164 a.The precursor sequence is 126 bp in length and can be folded into a typical stem and loop structure.Its mature fragment(5'-UGGAGAAGCAGGGCACGUGCA-3')is located on the 5' stem structure of the precursor structure.The mature sequence of Csn-miR164 a is highly conserved and is consistent with the mature sequence of miR164 family other plants.The Gi319875685 mRNA sequence was obtained from the tea plant transcriptome and was specifically amplified to obtain the ORF sequence,which is 912 bp in length and encode a protein of 303 amino acids.This sequence has a homology of 99% with a NAC1 gene of tea plant registered by NCBI(accession number: JN857066.1),and therefore,is named CsNAC1.There is a typical NAM domain at the N-terminus and a Csn-miR164 a recognition site at the C-terminus of the CsNAC1 protein.(2)Analysis of the Expression of miR164 a and Its Target Gene CsNAC1 of teaThe analysis of temporal and spatial expression characteristics showed that Csn-miR164 a had the highest expression in buds and the lowest in the seventh leaf,which was negatively correlated with CsNAC1.This indicated that Csn-miR164 a is involved in the growth of tea plant by negatively regulating CsNAC1.The high temperature treatment of tea plants showed that Csn-miR164 a was negatively correlated with CsNAC1,suggesting that Csn-miR164 a may increase the temperature tolerance of tea plants by negatively regulating CsNAC1 under high temperature stress.The expression characteristics of two tea cultivars with low temperature and drought treated respectively indicated that Csn-miR164 a was negatively regulated with CsNAC1,and it was induced to a greater extent in Shuchazao than Yinghong 9,presumably it is related to differences in cold resistance of different tea varieties.(3)Functional Analysis of miR164aTreatment of tobacco plants transformed with pBI121-Csn-MIR164 a with PEG6000 mimicking drought stress showed that the leaf osmotic potential of tobacco leaves is significantly reduced compared to transgenic tobacco.The ability of wild-type tobacco to regulate drought stress is greater than that of transgenic tobacco.Treatment of tobacco plants transformed with pBI121-Csn-MIR164 a gene under low temperature stress significantly decreased the Fv/Fm values of transgenic plants.The MDA content of transgenic tobacco was significantly higher than that of wild type tobacco,but its adaptability to low temperature environment was decreased.Wild type and transformed tobacco plants were subjected to low temperature stress at 4 °C,and the expression of endogenous and exogenous miR164 a and target genes were detected.The results showed that Csn-miR164 a was overexpressed in tobacco driven by the CAM35 S promoter and responded to low temperature stress by negatively regulating the expression of its target gene NtNAC100.In conclusion,miR164 a inhibits plant resistance to drought and low temperature stress by negatively regulating its target genes in transgenic pBI121-Csn-MIR164 a tobacco plants.